This invention relates to the field of skin lightening. Specifically, this invention relates to novel skin lightening compositions and methods of using the subject compositions to achieve skin lightening in mammals.
Skin lightening is an important skin care need, especially in the Asian population. This includes overall lightening of basal skin tone and hyperpigmented lesions. It is generally known that conditions which result in defective or missing tyrosinase, an enzyme involved in the formation of melanin lead to a loss of pigmentation, e.g. albinism. Conversely, it is known that inhibition of tyrosinase may likely lead to skin lightening via inhibition of melanogenesis. See King, R. A. and C. G. Summers, Dermatologic Clinics, Vol. 6 pp. 217-227 (1988).
Tyrosinase is present within the melanosomes in epidermal melanocytes and catalyzes the committed step in the formation of melanin from tyrosine. See Goldsmith, L. A., Physiologyy, Biochemistry, and Molecular Biology of the Skin, Oxford University Press, pp. 873-903 (N.Y. 1991). Tyrosinase catalyzes the hydroxylation of tyrosine (as a tyrosine hydroxylase) and the oxidation of DOPA to DOPAquinone (as DOPA oxidase): 
Binding of an inhibitor to the active site of tyrosinase results in decreased melanin formation. See generally Prota, G. Melanins and Melanogenesis, Academic Press, Inc., (San Diego 1992). The art has produced certain tyrosinase inhibitors. However, it is well recognized in the art that any active in any composition, especially when used for topical application (whether for pharmaceutical or cosmetic purposes) must be efficacious, bioavailable, stable when exposed to light, air or to the skin. Should the product be unstable, the breakdown products of the active must be innocuous.
Currently, there are several tyrosinase inhibitors in the marketplace, including hydroquinone, kojic acid and arbutin. However, there are disadvantages to each of these products.
For example, kojic acid and arbutin are marginal tyrosinase inhibitors and also are not very bioavailable, thus they have marginal efficacy.
Another example, hydroquinone, is oxidized by air, light and tyrosinase itself. These oxidized products of hydroquinone have been implicated in skin irritation (and perhaps cytotoxicity) and in pigmentation rebound (i.e. initial lightening followed by darkening).
Therefore there is a need to provide a more effective skin lightening agent which is more efficacious than kojic acid or arbutin. In addition, there is a need to provide a stable tyrosinase inhibitor, which is resistant to oxidation from light, air, and tyrosinase, thus avoids the formation of by-products which can lead to skin irritation. The advantages of these more bioavailable and efficacious inhibitors are a noticeable lightening benefit with a lack of skin irritation. Other likely benefits will include ease of use, improved shelf life and decreased frequency of application. It is the object of this invention to provide such compounds and compositions.
This invention relates to compounds and compositions which achieve skin lightening in mammals and to their methods of use. These compounds and compositions provide compositions that have stability against oxidation and provide a stability advantage over many existing compositions. In addition, we have found that these compounds and compositions inhibit tyrosinase better than many prior art compounds and compositions and also are more bioavailable, thus they are more efficacious than the prior art.
Specifically, this invention relates to compositions and compounds for lightening skin having the structure: 
wherein:
(i) each X is, independently, selected from the group consisting of halo, C1-C10 alkyl, C1-C10 substituted alkyl, substituted or unsubstituted phenyl, substituted or unsubstituted naphthyl, OR, OCOR, COR, CONRR, COOR, CN, SR, SOR, SO2R, SO3R and NRR, wherein X is other than hydroxy, amino and thio, if this X is attached ortho to the phenol hydroxy;
(ii) m is an integer from 0 to 4;
(iii) each Rxe2x80x2 and each Rxe2x80x3 is, independently, selected from the group consisting of hydrogen, halo, C1-C10 alkyl, C1-C10 substituted alkyl, substituted or unsubstituted phenyl, substituted or unsubstituted naphthyl, OR, OCOR, OCRROR, COR, CR(OR)OR, CONRR, COOR, CRROR, CN, SR, and NRR; wherein halo, when it appears, is other than geminal to a hydroxy, NH2, or SH; wherein up to two Rxe2x80x2 and Rxe2x80x3 are other than hydrogen;
(iv) Rxe2x80x2xe2x80x3 is C1-C10 alkyl or C1-C10 substituted alkyl;
(v) each R is, independently, selected from the group consisting of hydrogen, C1-C10 alkyl, C1-C10 substituted alkyl, substituted or unsubstituted phenyl, and substituted or unsubstituted naphthyl;
(vi) n is an integer from 1 to about 5, wherein at least one carbon in (C)n has other than alkyl or hydroxy as a substituent, adjacent to Z has other than amino, SH, CN or hydroxy as Rxe2x80x2;
(vii) Z is selected from the group consisting of O, NR, S, SO, SO2, PO2R and POR;
(viii) wherein any carbon, when disubstituted, having as one substituent selected from the group consisting of hydroxy, amino, cyano and thiol, has the other substituent selected from the group consisting of hydrogen, C1-C10 alkyl, substituted or unsubstituted phenyl, and substituted or unsubstituted naphthyl, whether this substituent is Rxe2x80x2 or Rxe2x80x3.
Specifically included in this invention are pharmaceutically acceptable salts of these compounds, stereoisomers and enantiomers thereof free from or mixed with other enantiomers or stereoisomers and such compounds in compositions with a pharmaceutically-acceptable carrier thereof.
This invention further relates to methods of lightening skin in mammals by administering to the skin of a mammal a composition comprising a safe and effective amount of a subject skin lightening active.
We have unexpectedly found that the compounds and compositions of this invention lighten skin in mammals. Furthermore, we have unexpectedly found that these compounds have improved stability toward oxidation, and are more bioavailable and efficacious than the prior art.
This invention is not limited to any particular mechanism of action, but is believed to operate by the inhibition of tyrosinase, an enzyme crucial for the formation of melanin. In this mechanism, the bioavailablity of the active compound and its inhibition of tyrosinase are predictive of efficacy.
As used herein, xe2x80x9calkylxe2x80x9d means carbon-containing chains which may be straight, branched or cyclic; substituted or unsubstituted; saturated, monounsaturated (i.e. one double or triple bond in the carbon chain), or polyunsaturated (i.e. two or more double bonds in the carbon chain, two or more triple bonds in the carbon chain, one or more double and one or more triple bonds in the carbon chain). Unless otherwise indicated, alkyl are preferably as follows. Preferred alkyl are straight or branched chain, more preferably straight chain. Preferred alkyl are mono-, di-, or tri-substituted, or unsubstituted, most preferably unsubstituted. Preferred alkyl are saturated or monounsaturated and, if so, preferably with a double bond; more preferably alkyl are saturated. Preferred alkyl are C1-C10, more preferably C1-C4, also more preferably methyl, ethyl and t-butyl, more preferably still methyl and ethyl, most preferably methyl.
Thus the term xe2x80x9csubstituted alkylxe2x80x9d is included in the definition of alkyl. Preferred alkyl substituents (i.e. substitution on alkyls) include halo, aryl, amino, hydroxy, alkoxy, cyano, nitro, amino (including mono- and disubstituted amino) thiol and substituted thiol and trifluoromethyl. More preferred alkyl substituents are halo and aryl. Thus xe2x80x9chaloalkylxe2x80x9d is included in xe2x80x9calkylxe2x80x9d and includes, but is not limited to, trifluoromethyl, 1,1,1-trifluoroethyl, 1-chloroethylxe2x80x2, 3-chloropentyl, bromomethyl and the like.
As used herein the term xe2x80x9calkoxyxe2x80x9d includes the above described alkyl radicals attached to the molecule via oxygen. Thus alkyl not only includes the C1-C10 alkyloxy, but also includes species such as methylenedioxy, ethylenedioxy and other similarly bifunctional, or multifunctional alkoxy substituents. These multifunctional substituents can be attached to various places in the molecule and thus form bridged species. For example, species such as dioxolanes, dioxanes and the like are specifically contemplated.
As used herein xe2x80x9chaloxe2x80x9d means F, Cl, Br, and I. Preferred xe2x80x9chalosxe2x80x9d are F, Cl, and Br, more preferably F and Cl, most preferably F.
As used herein, xe2x80x9carylxe2x80x9d means aromatic rings which may be unsubstituted or substituted. Preferred aryl are phenyl or naphthyl, especially phenyl. Preferred aryl are mono-, di-, tri-substituted or unsubstituted; more preferred aryl are monosubstituted or unsubstituted, most preferred being unsubstituted. Preferred aryl substituents include alkyl, halo, amino, hydroxy, alkoxy, cyano, nitro and trifluoromethyl. More preferred aryl substituents are alkyl and halo. The most preferred aryl is unsubstituted and thus is phenyl.
As used herein, the naming of any element, atom or radical contemplates all isotopes thereof. Therefore, hydrogen includes deuterium, and tritium and similarly hydro, includes deutero, and the like.
As used herein, xe2x80x9cpharmaceutically-acceptable saltsxe2x80x9d include Na+, K+, Ca++, Mg++, Al2(OH)5+, NH4+, (HOCH2CH2)3NH+, (CH2CH2)3NH+, (CH3CH2)4N+, C12H25(CH3)3N+ and C12H25(C5H4N)3N+ and the like. It is understood from this definition that some salts may include surfactants as the counterion. Preferred salts include Na+, K+, NH4+, and (HOCH2CH2)3NH+. More preferred salts include Na+ and NH4+.
As used herein, xe2x80x9ctopical applicationxe2x80x9d means directly laying on or spreading on outer skin.
As used herein, xe2x80x9cpharmaceutically-acceptablexe2x80x9d means that salts, drugs, medicaments or inert ingredients which the term describes are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, incompatibility, instability, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio.
As used herein, xe2x80x9csafe and effective amountxe2x80x9d means an amount of compound or composition sufficient to significantly induce a positive modification in the condition to be treated, but low enough to avoid serious side effects (at a reasonable benefit/risk ratio), within the scope of sound medical judgment. The safe and effective amount of the compound or composition will vary with the particular condition being treated, the age and physical condition of the patient being treated, the severity of the condition, the duration of the treatment, the nature of concurrent therapy, the specific compound or composition employed, the particular pharmaceutically acceptable carrier utilized, and like factors within the knowledge and expertise of the attending physician.
As used herein xe2x80x9chyperpigmented lesionxe2x80x9d means a localized region having high melanin content. Examples of these include, but are not limited to age spots, melasma, chloasma, freckles, post inflammatory hyperpigmentation, sun-induced pigmented blemishes and the like.
As used herein, xe2x80x9cskin lighteningxe2x80x9d means decreasing melanin in skin, including one or more of; overall lightening of basal skin tone, lightening of hyperpigmented lesions including age spots, melasma, chloasma, freckles, post inflammatory hyperpigmentation or sun-induced pigmented blemishes.
As used herein, xe2x80x9cskin lightening agentxe2x80x9d means an active agent, or a pharmaceutically-acceptable salt thereof, as defined herein below.
As used herein, all percentages are by weight unless otherwise specified.
Simple chemical transformations which are conventional and well known to those skilled in the art of chemistry can be used for effecting changes in functional groups in the compounds of the invention. For example, acylation of hydroxy- or amino-substituted species to prepare the corresponding esters or amides, respectively; preparation of ethers, including methyl and benzyl ethers, or cleavage of methyl or benzyl ethers to produce the corresponding alcohols or phenols; and hydrolysis of esters or amides to produce the corresponding acids, alcohols or amines; aromatic substitution, including halogenation of aromatic rings, etc.; oxidation of alcohols to ketones, acids or aldehydes; and other reactions, as desired, can be carried out.
Active Agent
This invention involves the lightening of mammalian skin by administering to the skin a safe and effective amount of a compound having the structure: 
or a pharmaceutically-acceptable salt thereof.
In the above structures each X is, independently, selected from the group consisting of halo, alkyl, aryl, OR, OCOR, COR, CONRR, COOR, CN, SR, SOR, SO3R, SO2R and NRR; each X is preferably independently selected from the group consisting of halo, alkyl, haloalkyl, substituted alkyl, OR and OCOR; more preferably from the group consisting of F, Cl, Br, methyl, OH, OCH3 and OCOCH3.
In addition, where X is SH, NH2 or OH and is attached to the carbon ortho to the phenolic hydroxy, and such substituents render the molecule more prone to oxidation (a problem common to some prior art compounds) and thus species with these substituents when in this arrangement are not contemplated to be part of the invention, if they result in an unstable active.
In the above structures, each R is, independently, selected from the group consisting of hydrogen, alkyl, substituted alkyl and aryl; preferably hydrogen or alkyl.
In the above structures, m is an integer from 0 to 4; preferably 0 to 2; more preferably 0 or 1. Of course, when m is 0, then the aryl ring is unsubstituted.
In the above structures, each Rxe2x80x3 and Rxe2x80x2 is, independently, selected from the group consisting of hydrogen, halo, alkyl, substituted alkyl, aryl, OR, OCOR, OCRROR, COR, CONRR, CRROR, COOR, SR, NRR, and CN.
Rxe2x80x2 is preferably from the group consisting of hydrogen, halo, haloalkyl, aryl, and alkyl. In compounds of formula I, Rxe2x80x2 is more preferably C1 to C3 alkyl, hydroxy, halo, cyano or hydrogen, and still more preferably H, F, Cl, Br or methyl. In compounds of formula II, Rxe2x80x2 is preferably hydrogen or alkyl, more preferably hydrogen.
Rxe2x80x3 is preferably from the group consisting of hydrogen, halo, alkyl, haloalkyl, substituted alkyl, OR and OCOR; in compounds of formula I, more preferably from the group consisting of H, F, Cl, Br, methyl, OH, OCH3 and OCOCH3; in compounds of formula II, Rxe2x80x3 is preferably Cl to C3 alkyl or C1 to C3 alkyl substituted with methoxy, acetoxy, hydroxy, chloro, fluoro, or bromo.
For compounds of formula I; less than about four Rxe2x80x2 and Rxe2x80x3 are other than hydrogen, preferably less than three, most preferably up to two. In this case the most preferred substituents are hydroxy, halo, cyano and alkyl. However, in this case hydroxy, thiol cyano or any other substituents which will render the molecule unstable, these are not attached to the same carbon as Z. It is preferred that Rxe2x80x2 is alkyl, substituted alkyl, alkoxy or hydrogen for Rxe2x80x2 attached to the carbon adjacent to Z in the ring.
Thus it is apparent that certain radicals are not intended to be arranged to describe an inherently unstable molecule. The skilled artisan will immediately recognize that, certain substituents are specified so as not to appear in certain specific arrangements for this reason. For example, where any carbon has geminal hydroxy substituents, or a hydroxy and NH2, or hydroxy and SH, or geminal SH or geminal NH2, and such compounds are inherently unstable, these compounds and their compositions are not contemplated to be part of the invention. Likewise, where alpha-halo hydroxy and the like render the species inherently unstable, this species is not contemplated to be a part of the invention.
However, bearing these considerations in mind, it is understood by the skilled artisan that species are not inherently unstable (i.e. are stable) if they remain reasonably resistant to degradation in a composition on the shelf and in delivery or application.
In the above structures, n is an integer from 1 to 5; preferably 2 or 3.
In the above structures, Z is selected from the group consisting of O, NR, S, SO, SO2; POR and PO2R; more preferably O or S.
In the above structures, Rxe2x80x2xe2x80x3 is alkyl or substituted alkyl; preferably unsubstituted alkyl or alkyl substituted with halo, aryl, COR, CONRR, hydroxy, or alkoxy; more preferably methyl, CF3 or C1-C4 alkyl substituted with F, Cl, Br, CF3 or OCH3.
The compounds of the invention are useful both in the form of the active itself and the form of salts of the active (i.e., cosmetically or pharmaceutically acceptable salts), and both forms are within the purview of the invention. The salts may be in some cases a more convenient form for use, and in practice the use of the salt form inherently amounts to the use of the active itself. In addition to convenience, the salt may aid in dissolution, topical delivery and the like of the active. The preferred moieties which can be used to prepare the salts include those which produce, when combined with the free base, pharmaceutically or cosmetically acceptable salts, that is, salts whose counterions are relatively innocuous to the animal organism in common doses of the salts so that the beneficial properties inherent in the free form are not vitiated by side effects ascribable to the counterion. Appropriate acceptable salts within the scope of the invention are those derived from other mineral acids and organic acids or mineral bases and organic bases, depending upon the pKa of the active. Here bases, as described above, are preferred in preparation of the salt. These salts are prepared in conventional ways, for example, by dissolving the free molecule in aqueous alcohol solution containing the appropriate counterion or counterion precursor (e.g. an unprotonated base or protonated acid; which are not ionized and thus soluble) and then isolating the resulting salt by evaporating the solution, or by reacting the free molecule and a counterion or counterion precursor in an organic solvent, in which case the salt separates directly, is precipitated with a second organic solvent, or can be obtained by concentration of the solution; other methods exist. All salts are useful as sources of the free form of the active, even if the particular salt per se is desired only as an intermediate product, as, for example, when the salt is formed only for purposes of purification, identification, or when it is used as an intermediate in preparing a medicinally acceptable salt by ion exchange procedure, or as a means to purify an enantiomer or stereoisomer.
It is recognized that the compounds of the invention can exist as stereoisomers, and as such the description of the compounds includes all stereoisomers and mixtures thereof. Furthermore, it is understood that the skilled practitioner can selectively prepare the desired stereoisomer, using selective synthetic methods. These methods include (but are not limited to) temperature control (to prepare kinetic vs thermodynamically favored products), selective catalysts and/or chiral solvents (which encourage the preparation of one stereoisomer over another, even in prochiral molecules), chiral auxilliaries, the choice of specific reactions, and the like. It is also recognized that methodologies exist for the separation of chiral mixtures including (but not limited to) preparation of a salt using a chiral counterion (for example, tartrate and other chiral anions or cations), use of a chiral solvent (for example sec-butanol), use of stereoselective chromatography and the like. Such selection of stereoesomers is often advantageous as one stereoisomer can be more active than another. Thus it is within the scope of the invention to have mixtures of stereoisomers as well as one or more stereoisomer(s) substantially free of the other stereoisomer(s). For example, it is known that one stereoisomer of one of the compounds of this invention inhibits tyrosinase better than the other stereoisomer, and thus it is sensible and contemplated that a practitioner might prefer to treat the mammal in need of treatment with the stereoisomer which inhibits tyrosinase better, all other properties being equal.
In addition, it is recognized that the compounds of the invention exist as enantiomers. Since the same considerations apply to enantiomers as stereoisomers, it is expected that a practitioner might prefer to treat the mammal in need of treatment with the enantiomer which inhibits tyrosinase better, all other properties being equal. Thus it is within the scope of the invention to have a mixture of enantiomers as well as one enantiomer substantially free of the other enantiomer.
It is also apparent that making minor changes in the reaction""s conditions leading to the desired product is within the purview of the skilled artisan in organic chemistry. For example, adjusting temperature/pressure of a reaction, adjusting workup conditions to maximize recovery of the desired material, increased time for reaction and the like, are strategies employed to increase yield and are often not crucial to the successful synthesis of the desired molecule. In addition, the selection of preferred reactants in any given reaction is generally a matter of selection, for example, the choice of one acid over another, when used to protonate a reactant, is neither crucial to the successful synthesis, nor outside the purview of the skilled artisan.
In the methods of preparing compounds of the invention below, or elsewhere, any carbon with clearly undesignated substitution has the appropriate number of hydrogens.
Compounds with cyclic heterocycles, wherein Z is oxygen are conveniently prepared by the enol-ether synthesis as illustrated below; 
In addition, variations in stereochemistry can be effected by utilizing various synthetic methods. For example an xcex1,xcex2 epoxide of pyran, prepared in situ, can be reacted (or trapped) under electrophilic conditions to prepare a trans-hydroxy compound. The trans-oxycyclic actives having hydroxy, methoxy, acyloxy and the like can be prepared by analogy to the following method; 
The analogous cis-oxycylic compounds are prepared by oxidation/reduction of the trans compound. It is contemplated that the selective synthesis is performed and prescribed by the desired end product to be used as an active. Of course, rings of other types and sizes can be manipulated using similar methods with different starting materials.
Preparation of the compounds with heterocycles wherein Z is NR are prepared by known methods (Cf. Hanessian, S. (1965) Chemistry and Industry 1296-1297; Heathcock, C. H., Norman, M. H., and Dickman, D. A. (1990) J. Org. Chem. 55, 798-811; Shono, T., Matsumura, Y., Onomura, O., and Yamada, Y. (1987) Tetrahedron Letters 28, 4073-4074) and by methods analagous to those of compounds wherein Z is O.
Compounds with heterocycles wherein Z is sulfur can be prepared by analogy to the method of Giovani, E. Napolitano, E., and Pelosi, P. (1993) Gazzetta Chimica Italiana 123, 257-260. The resulting thioethers can then be oxidized to sulfoxides and subsequently to sulfones using known oxidizing reagents, such as peroxides, PCC, KMnO4 and the like. 
Compounds with Zxe2x95x90PO2R and POR are prepared by analogy to known chemistries (Cf. Inokawa, S., Kitagawa, H., Kuniaki, S., Hiroshi, Y., and Ogata, Y. (1973) Carbohydrate Research 30, 127-132;Hanaya, T., Nobuyuki, S. Yamamoto, H., Armour, M-A., and Hogg, A. M. (1990) Bull. Chem. Soc. Jpn. 63, 421-427) Starting materials chosen to prepare these compounds of the invention, are known, or are prepared by known methods. Once the materials are chosen, such compounds are prepared and manipulated by known chemistries.
Compounds with acyclic groups are prepared by several methods; preferred methods for preparing compounds with Rxe2x80x2 as hydrogen and Z as O or S include; 
wherein ORx is a group that can be displaced by an anion, B is a blocking group, Y is a leaving group and Ph is aryl, and where each Rp independently is a precursor to Rxe2x80x3, such that Rxe2x80x3 is CRpRp.
Other vinyl ethers and thio vinyl ethers are prepared by several known methods, for example, by reactions of ketones, or aldehydes with Wittig reagents and the like. Many vinyl ethers and thioethers are known or commercially available, and these are also expected to be useful in the final reaction step above thus producing the desired active product.
Compounds wherein Rxe2x80x3 is methyl are prepared by the following scheme as well; 
where Y is a leaving group, including tosyl, halo, and the like.
Where Z is O, NR or S, a method analogous to the following is employed to prepare the compounds of the invention: 
This synthesis is useful for nearly any Rxe2x80x2xe2x80x3. Where Rxe2x80x3 is hydroxymethylene a method analogous to the following scheme is preferred; 
Other epoxidation agents can be used in the first step, including m-chloroperbenzoic acid (MCPBA) and the like. Where Z is O or NR, compounds are prepared by analogy to the preceding scheme; 
Where RRxe2x80x2xe2x80x3NH is any secondary amine in the presence of a strong base.
Of course, blocking and unblocking of phenolic hydroxy (using groups designated B above) and the like are done by art recognized methods, such as using the benzyl group, and later reducing the group off with palladium on carbon and the like.
The structures of the compounds of the invention are established by the mode of synthesis, by elemental analysis, and by infrared, nuclear magnetic resonance, or mass spectroscopy. The course of the reactions and the identity and homogeneity of the products are often assessed by thin layer chromatography (TLC) and high pressure liquid chromatography (HPLC) and melting point.
Once prepared, the tyrosinase inhibition activity of the active compound is determined by standard enzyme kinetic methods. The in vitro determination of a Ki value is a well accepted parameter to judge the strength of a compound""s inhibition of an enzyme. The art describes assays which show inhibition of tyrosinase by other molecules, but these assays are inherently insensitive and typically inaccurate.
An improved tyrosinase assay combining the resolution of HPLC and the sensitivity of fluorescence detection is developed to provide a more reproducible and sensitive measurement of tyrosinase activity and inhibition. In this assay, concentrations of tyrosine, DOPA, and tyrosinase are optimized to determine the kinetic parameters of tyrosinase. The assay measures conversion rates of tyrosine to 3,4-dihydroxyphenylalanine (DOPA) catalyzed by tyrosinase (i.e. tyrosine hydroxylase activity). HPLC separates tyrosine from other assay components to provide reproducible substrate quantitation. HPLC-fluorescence detection provides quantitation of concentrations of tyrosine to as low as 0.1 xcexcM tyrosine and changes in concentration smaller than 0.1 xcexcM. This improved assay reliably determined the strength of inhibition (Ki) as well as the type of inhibition (i.e. competitive vs. noncompetitive inhibition) of enzyme inhibitors with excellent reproducibility and sensitivity. This assay allows for the quantitative comparison of known tyrosinase inhibitors and novel compounds.
Tyrosinase is commercially obtained. Kinetic assays to determine the strength (Ki) and type of inhibition of inhibitors are performed as described and summarized. Inhibitors at varying concentrations (from nanomolar to millimolar) are incubated in the presence of tyrosine (1-50 xcexcM) and tyrosinase (2 U/mL) in 100 mM of pH 7.0 MOPS buffer containing 0.5 xcexcM DOPA. The samples are analyzed at times 0, 6, 12, 18, and 24 min by HPLC chromatography (using a Supelco cyano analytical column) for the depletion of tyrosine. A fluorescence detector (xcexexcit=260 nm xcexemis=305 nm) is used to detect tyrosine. The Ki values and type of inhibition are determined from graphic analysis of data using the accepted Lineweaver-Burke and Dixon plots. The inhibition values for preferred compounds (i.e. the Ki values) are determined by standard methods and showed good to excellent tyrosinase inhibition.
The actives are tested for oxidation by tyrosinase, and none are appreciably oxidized by tyrosinase.
Actives are tested in solution and composition for stability, including stability toward light, air, and water. None of the actives tested showed appreciable oxidation by air or light. Based upon these results, it is shown that stable formulations could be prepared where the active is not appreciably oxidized by air or light.
Skin penetration values (Jmax) are predicted for each example compound from the flux equation as described by Kasting, et al., 1992. Jmax is defined as the flux of a moderately lipophilic solute across the skin from vehicle. The compound""s parameters (e.g., melting point, molecular weight, clog p (calculated partition coefficient)) are used to calculate the flux value as written:
Jmax=(Dlip/hlip)*Slip 
where Jmax is the maximum flux through the barrier (xcexcg/cm2/h)
hlip is the effective thickness of the stratum corneum lipid barrier (cm)
Dlip is the diffusion coefficient of the drug in this barrier (cm2/h)
Slip is the solubility of the drug in this barrier (xcexcg/cm3)
This model predicts penetration of compounds through skin. In fact, compounds of the invention are predicted to be more bioavailable than arbutin and kojic acid based on this parameter (Jmax). Thus the actives are predicted to be efficacious without the common drawbacks associated with prior art compounds such as oxidation, lack of bioavailability, or poor tyrosinase inhibition. Preferred compounds have Jmax of 2 xcexcg/cm2/h or greater, when used topically.
Preferred compounds (which are predicted to penetrate skin well and displayed excellent tyrosinase inhibition) are tested in the pigmented guinea pig, an art accepted model for skin lightening efficacy, to determine their in vivo efficacy in a composition. On each guinea pig from two to six treatment sites (typically 16 cm2 each) are treated topically with compositions containing preferred compounds (100 xcexcL of 0.1-3% active, 5xc3x97 per week for up to 6 weeks). The animals are visually and instrumentally graded with a Minolta Chromameter (CR-300) for erythema (i.e., a redness scale using xe2x80x9caxe2x80x9d values) and pigmentation (i.e., a lightness scale using xe2x80x9cLxe2x80x9d values). Each week the treatment sites on the animals are also photographed. By both visual and instrumental methods, the compounds tested in vivo lightened skin, without appreciable irritation or pigmentation rebound.
Based on results above it is believed that skin lightening compositions of this invention preferably comprise from about 0.001% to about 10% of a subject active compound in a topical composition, more preferably from about 0.01% to about 8%, more preferably still from about 0.1% to about 5%, most preferably from about 0.5% to about 5% of an active compound. Use of subject compositions comprising at least 5% of an active is preferred for lightening of hyperpigmented lesions and other areas where substantial lightening is desired.
Preferred compounds of the invention include; 
The most preferred compounds of this invention include; 4-[(tetrahydro-2H-pyran-2-yl)oxy]phenol; 4-[(tetrahydro-2H-thiopyran-2-yl)oxy]phenol; 2-fluoro-4-[(tetrahydro-2H-pyran-2-yl)oxy]phenol; 4-[(tetrahydrofuran-2-yl)oxy]phenol; and 4-[(1-ethoxyethyl)oxy]phenol.
The compounds of the invention are prepared by conventional methods using known starting materials. However, it is readily apparent to the skilled practitioner in organic chemistry that certain starting materials may be novel, but are made by methods which are known in the art. Moreover, the skilled artisan will recognize that some reactions are best performed by blocking functional groups on the reactants that may engage in undesirable side reactions. The recognition of the possibility of such reactions, the selection of blocking moieties to protect functional groups and the optimization of reactions with or without such groups is well within the purview of the skilled artisan.
The following examples are provided to further illustrate the invention, while not limiting the invention hereto.
The following examples are illustrative of the preparation of compounds useful in this invention.